REVIEW ARTICLE

published: 12 October 2012

doi: 10.3389/fpls.2012.00227

Evolution and structural diversification of PILS putative

auxin carriers in plants
Elena Feraru 1 , Stanislav Vosolsobě 2 , Mugurel I. Feraru 1 , Jan Petrášek 2,3 and Jürgen Kleine-Vehn 1 *
1
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria
2
Department of Faculty of Science, Experimental Plant Biology, Charles University, Prague, Czech Republic
3
Institute of Experimental Botany of the Academy of Sciences of the Czech Republic, Prague, Czech Republic

Edited by:
Markus Geisler, University of
Fribourg, Switzerland
Reviewed by:
Viktor Zarsky, Charles University,
Czech Republic
Eric M. Kramer, Bard College at
Simon’s Rock, USA
*Correspondence:
Jürgen Kleine-Vehn, Department of
Applied Genetics and Cell Biology,
University of Natural Resources and
Life Sciences, Muthgasse 18, A-1190
Vienna, Austria.
e-mail: juergen.kleine-vehn@
boku.ac.at
The phytohormone auxin contributes to virtually every aspect of the plant development.
The spatiotemporal distribution of auxin depends on a complex interplay between auxin
metabolism and intercellular auxin transport. Intracellular auxin compartmentalization pro-
vides another link between auxin transport processes and auxin metabolism.The PIN-LIKES
(PILS) putative auxin carriers localize to the endoplasmic reticulum (ER) and contribute to
cellular auxin homeostasis. PILS proteins regulate intracellular auxin accumulation, the rate
of auxin conjugation and, subsequently, affect nuclear auxin signaling. Here, we investigate
sequence diversification of the PILS family in Arabidopsis thaliana and provide insights into
the evolution of these novel putative auxin carriers in plants. Our data suggest that PILS
proteins are conserved throughout the plant lineage and expanded during higher plant
evolution. PILS proteins diversified early during plant evolution into three clades. Besides
the ancient Clade I encompassing non-land plant species, PILS proteins evolved into two
clades. The diversification of Clade II and Clade III occurred already at the level of non-
vascular plant evolution and, hence, both clades contain vascular and non-vascular plant
species. Nevertheless, Clade III contains fewer non- and increased numbers of vascular
plants, indicating higher importance of Clade III for vascular plant evolution. Notably, PILS
proteins are distinct and appear evolutionarily older than the prominent PIN-FORMED auxin
carriers. Moreover, we revealed particular PILS sequence divergence in Arabidopsis and
assume that these alterations could contribute to distinct gene regulations and protein
functions.
Keywords: PILS proteins, auxin, evolution, phylogeny, auxin metabolism, auxin homeostasis

INTRODUCTION
Plant development is particularly flexible due to its postembry-
onic growth behavior, allowing individual adjustment of the body
plan according to the environment (Finet and Jaillais, 2012).
The phytohormone auxin is crucial for these adaptive responses
and, hence, has drawn enormous research attention (Teale et al.,
2008). The importance of auxin for plant development seems
to be also reflected in the complex regulation of auxin percep-
tion and its spatiotemporal distribution (Vanneste and Friml,
2009). Up to date three auxin receptor classes have been sug-
gested to jointly regulate auxin-signaling output. Most auxin
responses have been assigned to the F-box proteins TRANS-
PORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX
(TIR1/AFB). Auxin binding to the co-receptors TIR1/AFB and
the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) will initiate the
proteasome-dependent degradation of the transcriptional repres-
sors Aux/IAAs. The subsequent release of AUXIN RESPONSE
FACTOR (ARF) transcription factors eventually leads to the tran-
scriptional reprogramming of the respective cell (Leyser, 2006;
Chapman and Estelle, 2009). Another F-box protein S-PHASE
KINASE-ASSOCIATED PROTEIN 2A (SKP2A) also binds to
auxin and might contribute to auxin-dependent modulation of
the cell cycle (Jurado et al., 2010). Rapid and non-genomic auxin
effects appear to be mainly perceived by the AUXIN BINDING
PROTEIN1 (ABP1; Jones and Venis, 1989; Robert et al., 2010;
Xu et al., 2010). However, ABP1 action might also affect auxin-
dependent gene transcription and cell cycle regulation (Braun
et al., 2008; Tromas et al., 2009).
Beside the complex cell type-dependent regulation of auxin sig-
naling, also auxin metabolism is multifaceted. Several redundant
auxin biosynthesis pathways determine auxin levels in various tis-
sues and the decay/inactivation of auxin is regulated via oxidation
or mostly reversible conjugation (Woodward and Bartel, 2005;
Ruiz Rosquete et al., 2012; Zhao, 2012). Auxin metabolism is highly
dynamic and has pronounced importance for the spatiotemporal
regulation of auxin.
Intercellular (polar) auxin transport also determines cellular
auxin levels (Zazímalová et al., 2010). The most prominent auxin
carriers are AUXIN-RESISTANT1/LIKE AUX1 (AUX/LAX) influx
carriers, ATP BINDING CASSETTE (ABC) auxin transporters of
a MULTIDRUG RESISTANCE (MDR) subfamily, and the PIN-
FORMED (PIN) auxin carriers (Bennett et al., 1996; Chen et al.,
1998; Gälweiler et al., 1998; Luschnig et al., 1998; Müller et al.,
1998; Utsuno et al., 1998; Geisler et al., 2005). PIN proteins have a

www.frontiersin.org October 2012 | Volume 3 | Article 227 | 1

Feraru et al.
particular developmental importance as their polar localization
at a given cell side determines the direction of the intercellu-
lar auxin flow (Wisniewska et al., 2006). PIN proteins can be
grouped into two subclasses according to the length of the central
hydrophilic loop. Canonical PIN1-type auxin efflux carriers have
a long loop, localize to the plasma membrane (PM) and perform
a rate-limiting function in cellular auxin efflux (Petrásek et al.,
2006). In contrast, PIN5 and PIN8 have a dramatically reduced
central hydrophilic loop, localize to the endoplasmic reticulum
(ER) and regulate intracellular auxin compartmentalization and
homeostasis (Mravec et al., 2009; Bosco et al., 2012; Ding et al.,
2012).
We have recently discovered a novel putative auxin carrier fam-
ily of seven members in Arabidopsis thaliana (Barbez et al., 2012)
and designated them as PIN-LIKES (PILS), because their predicted
protein topology is highly similar to the topology of the PIN pro-
teins. Similar to PIN proteins, PILS contain the so-called Interpro
auxin carrier domain, an in silico defined domain to predict auxin
transport function. Functional PILS5-GFP fusion proteins local-
ize to the ER and stimulate intracellular auxin accumulation in
plant and yeast cells (Barbez et al., 2012). PILS2 and PILS5 activ-
ity increases amide auxin conjugates, thereby reducing the free
auxin levels, and negatively affecting nuclear auxin signaling (Bar-
bez et al., 2012). Our current working model proposes that PILS2
and PILS5 proteins regulate auxin compartmentalization into the
ER lumen, where auxin might be the substrate for compartmen-
talized auxin metabolism (Figure 1). It needs to be experimentally
tested whether PILS proteins affect nuclear auxin signaling mainly
by limiting the excess of auxin to diffuse into the nucleus or by the
effect on presumably compartmentalized auxin conjugation. This
mode of action is reminiscent to auxin carrier PIN5 that has been
shown to regulate intracellular auxin homeostasis by modulating
auxin compartmentalization and metabolism at the ER (Mravec
et al., 2009). Further research will address whether the distinct
protein families have redundant and interchangeable function at
the ER.
PILS overexpression strongly distorts plant patterning and
development, while pils2 and pils5 loss of function mutants show
comparably weaker defects in plant growth regulation. Moderate
PILS5 gain and pils2pils5 loss of function phenotypes can be largely
explained by low and high auxin content, respectively. For exam-
ple, PILS5 overexpressors have reduced free auxin levels/signaling,
shorter hypocotyls and fewer lateral roots, while pils2pils5 dou-
ble mutants display higher free auxin levels, enhanced hypocotyl
growth and lateral rooting. In contrast, PILS5 gain andpils2pils5
loss of function leads to reduced and enhanced root growth,
which might be not related to the overall changes in auxin con-
tent, but could indicate a more specific PILS2 and PILS5 func-
tion in the cellular regulation of root growth (Barbez et al.,
2012).
The identification of PILS proteins and their role in auxin
homeostasis at the ER reveal the molecular complexity of intra-
cellular auxin compartmentalization and its eminent impor-
tance for the plant development. Here we present in silico
analysis to further reveal insight into the organization and
regulation of this novel family of putative auxin transport
facilitators.
Frontiers in Plant Science | Plant Traffic and Transport
PILS evolution and structural diversification
MATERIAL AND METHODS
SEQUENCE INFORMATION
Sequences were downloaded from PLAZA1 , NCBI2 by using
tblastx program (Altschul et al., 1997; nr/nt database, PILS and PIN
sequences from A. thaliana as queries) or Phytozome3 servers. The
information and the ID of the presented sequences can be found
in the Supplementary Data.
ONLINE SERVERS
The online available servers used to perform in silico analyses
of PILSes are found at: http://www.arabidopsis.org (chromosome
localization; alternative splicing), http://bioinformatics.psb.ugent.
be/plaza/ (intron/exon organization; sequence information),
www.genevestigator.com (expression), http://bar.utoronto.ca/efp/
cgi-bin/efpWeb.cgi (expression), http://www.enzim.hu/hmmtop/
index.php (prediction of protein topology), http://biophysics.
biol.uoa.gr/TMRPres2D (visual representation of transmembrane
protein models), http://weblogo.berkeley.edu/logo.cgi (sequence
logo), http://www.cbs.dtu.dk/services/NetPhos/ (prediction of
phosphorylation sites), http://www.cbs.dtu.dk/services/NetPhosK
(prediction of phosphorylation sites), http://www.ebi.ac.uk/Tools/
msa/clustalw2/ (multiple amino acid sequences alignment), http:
//blast.ncbi.nlm.nih.gov/ (sequence information), http://www.
phytozome.net/ (sequence information), http://www.r-project.
org/ (analysis of collinearity).
ANALYSIS OF COLLINEARITY
We investigated possible collinearity among A. thaliana PILS
genes by comparing 200 surrounding translated genes for each
PILS. The comparison was performed for pairs of PILS genes by
using blastp program (Altschul et al., 1997). The homology was
1 http://bioinformatics.psb.ugent.be/plaza/
2 http://www.ncbi.nlm.nih.gov/
3 http://www.phytozome.net/
FIGURE 1 | Model of PILS protein function in Arabidopsis thaliana.
PILS2 and PILS5 proteins localize to the ER and mediate intracellular
auxin accumulation. We hypothesize that PILS proteins are putative
auxin carriers that regulate the auxin transport from the cytosol into
the lumen of the ER (black arrow). PILS activity affects auxin
metabolism and might control the cytoplasmic availability of auxin
(adapted from Barbez et al., 2012).
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Feraru et al. PILS evolution and structural diversification

Table 1 | Size of PILS gene families in different plant species.

determined according to E-value from blast results. The analysis
was performed in R environment4 . Lineage Species Number of genes*

PHYLOGENETIC ANALYSIS Eukaryota

A multiple alignment was built by using Muscle in MEGA5 soft- Viridiplantae
ware (Tamura et al., 2011). Only the conserved domains were Chlorophyta Micromonas sp. 1
used and all positions with less than 80% site coverage were Micromonas pusilla 1
eliminated. The evolutionary history was inferred by using the Ostreococcus lucimarinus 1
Maximum Likelihood method based on the Whelan and Gold- Ostreococcus tauri 1
man (2001) + Freq. model with discrete Gamma distribution (five Chlamydomonas reinhardtii 2
categories, G parameter = 3.0640) for analysis of PILS amino acid Volvox carteri 2
sequences or on the Whelan and Goldman model with discrete Bryophyta Physcomitrella patens 5
Gamma distribution (five categories, G parameter = 2.9920) for Lycopodiophyta Selaginella moellendorffii 8
analysis of PIN-PILS dataset. The trees are drawn to scale, with Euphyllophyta
branch lengths measured in the number of substitutions per site. Monocots Oryza sativa japonica 6
The PILS analysis involved 42 amino acid sequences and 322 Oryza sativa indica 6
positions in the final dataset. The PIN-PILS analysis involved 67 Sorghum bicolor 7
amino acid sequences and 354 positions. Evolutionary analyses Brachypodium distachyon 8
were conducted in MEGA5 (Tamura et al., 2011). For the sequence Zea mays 10
alignments see Figures S2 and S3 in Supplementary Material. Dicots Carica papaya 4
Arabidopsis lyrata 6
RESULTS Arabidopsis thaliana 7
PHYLOGENY OF PILS PROTEINS Ricinus communis 7
Using available online tools, we previously showed that PILS pro- Fragaria vesca 8
teins are highly conserved among plant species (Barbez et al., Manihot esculenta 9
2012). To further investigate the evolution of PILS protein diver- Lotus japonicus 12
sification, we analyzed PILS protein sequences from all sequenced Medicago truncatula 13
taxa of Viridiplantae. The PILS family is present in all the 26 avail- Vitis vinifera 13
able sequenced genomes and is represented by 202 genes (Table 1; Malus domestica 14
Van Bel et al., 2012; confirmed by reciprocal blast, Altschul et al., Theobroma cacao 16
1997). PILS family obviously diversified in the different plant lin- Glycine max 17
eages (Table 1). Ancient species, such as algae (1–2), mosses (5), Populus trichocarpa 18
and spike mosses (8), have 1–8 PILS genes, while seed plants, such
*Gene information and sequences were retrieved from PLAZA platform (Van Bel
as Oryza (6), Zea (10), Medicago (13), or Populus (18), have 6 to
et al., 2012) and candidates were evaluated by reciprocal blasts (Altschul et al.,
18 PILS genes (Table 1). The steadily increasing number in seed
1997).
plants suggests that PILS genes have duplicated independently in
several plant lineages and indicate a more diversified function of
PILS proteins in higher plants. include these sequences in the phylogenetic analysis because they
To assess the evolutionary relationship among PILS proteins, we were incomplete (only EST fragments are currently available).
constructed phylogenetic trees with PILS sequences from selected The evolutionary Clade II and III already emerged early dur-
model organisms such as available green algae, Physcomitrella, ing non-vascular plant evolution and both contain PILS sequences
Selaginella, Picea, Brachypodium, Oryza, Medicago, Arabidopsis, from Embryophytes (land plants; Figure 2). The main lineages of
and Populus sequences (Figure 2; Figure S1 in Supplementary land plants are mosses, liverworts, hornworts, lycophytes, ferns,
Material; for sequence alignment see Figure S2 in Supplementary gymnosperms, and angiosperms. Clade II includes the well-
Material). The phylogenetic tree presented in Figure 2 shows that conserved PILS2- and PILS6-like subclades, including orthologs of
PILSes from Viridiplantae can be grouped into three evolutionary PILS2 and PILS6 from Physcomitrella, Selaginella, Brachypodium,
clades: Clade I, Clade II, and Clade III. or Oryza (Figure 2).
The available green algae genomes from the lineage Chlorophyta Clade III encompasses the PILS1/PILS3/PILS4- and PILS5/PILS7-
have a relatively low number of only one or two PILS genes per like subclades and displays particular expansion in higher seed
species. All these PILS algae orthologs cluster together and define plants (Figure 2; Figure S1 in Supplementary Material). Accord-
the Clade I that contains the so far oldest known PILS genes of ingly, this clade encompasses also most Brachypodium and Oryza
the Viridiplantae (Figure 2). We could also identify putative PILS orthologs (Figure 2; Figure S1 in Supplementary Material). Inter-
genes in the genomes of sequenced algae from lineage Strepto- estingly, one Physcomitrella and two Selaginella PILS sequences
phyta from which the land plants evolved. However, we did not are present at the root of the Clade III (Figure 2). The relatively
low number of moss and the relative over amount of higher plant
sequences in Clade III may suggest particular importance of this
4 http://www.r-project.org/ clade in vascular plant evolution.

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Feraru et al. PILS evolution and structural diversification

FIGURE 2 | Phylogeny of PILS proteins. The phylogenetic tree of PILS Likelihood molecular phylogenetic analysis was performed in MEGA5 (Tamura
proteins can be divided into three clades: Clade I (blue), Clade II (green), and et al., 2011) by using 42 amino acid PILS sequences from algae,
Clade III (red). The Arabidopsis PILSes are found in the Clade II and Clade III, Physcomitrella, Selaginella, Brachypodium, Oryza, and Arabidopsis as
while Clade I is represented only by PILS proteins from algae. The Maximum explained in the Materials and Methods.

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Feraru et al. PILS evolution and structural diversification

FIGURE 3 | Phylogeny of PILS and PIN proteins. PILS proteins are amino acid sequences from algae, Physcomitrella, Selaginella,
evolutionarily distinct of PIN proteins. Note the two separated Brachypodium, and Arabidopsis. For a better visualization the algae
subtrees. The Maximum Likelihood phylogenetic analysis was and sometimes the Physcomitrella and Selaginella branches were
performed in MEGA5 (Tamura et al., 2011) by using PILS and PIN collapsed.

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Feraru et al. PILS evolution and structural diversification

Our analysis reveals that PILS proteins are evolutionarily

conserved throughout plant evolution and might uncover the
versatile importance of compartmentalized auxin homeostasis
throughout the plant kingdom.

PILS PROTEINS ARE EVOLUTIONARILY DISTINCT OF PIN PROTEINS

The canonical PIN proteins act in the cellular efflux of auxin at the
plasma membrane, but the most ancient members of PIN proteins
(PIN5-type) localize to the ER and regulate the subcellular com-
partmentalization of auxin and auxin metabolism (Mravec et al.,
2009). Hence, both PILS and PIN5-like proteins localize to the ER
and regulate auxin homeostasis, presumably by mediating auxin
transport at the ER (Mravec et al., 2009; Barbez et al., 2012; Bosco
et al., 2012; Ding et al., 2012).
Next, we investigated the evolutionary relationship between
PILS and PIN proteins (Figure 3; for sequence alignment see
Figure S3 in Supplementary Material). The phylogenetic analysis
of PILS and PIN sequences from algae, moss, spikemoss, and sev-
eral Angiosperms revealed that PILSes and PINs form two distinct
phylogenetic clades (Figure 3). Although having a similar pre-
dicted protein structure and possibly similar function at the ER,
PIN and PILS proteins are evolutionarily distinct in plants. In con-
trast to PILSes, we could not find any PIN sequence in the genomes
of Chlorophyta algae, such as Chlamydomonas, Micromonas, Ostre-
ococcus, or Volvox. Notably, a truncated PIN sequence has been
found in the genome of Spirogyra (De Smet et al., 2011). These
findings indicate that PILS proteins are more conserved during
plant evolution and seem evolutionarily older than PIN pro-
teins. Therefore, we assume that the PILS proteins are central to
the evolution of intracellular auxin transport, which presumably
has preceded the evolution of PIN-dependent intercellular and
intracellular auxin transport.

PILS DIVERSIFICATION IN ARABIDOPSIS THALIANA

The seven Arabidopsis PILS genes are placed on chromosome 1,
2, and 5 (Figure 4). PILS1 to PILS4 are found on chromosome
1, PILS5 on chromosome 2, while PILS6 and PILS7 are both
placed at the ends of the chromosome 5 (Figure 4). PILS3 and
PILS4 are neighboring genes at the bottom arm of the chromo-
some 1 (Figure 4), indicating that PILS3 and PILS4 may resulted
from a gene duplication event. To investigate PILS paralogs in A.
thaliana we performed comparative sequence analysis of genes
that surround the seven PILS genes (Figure 5). Rows of 200
translated genes surrounding each of the seven PILS genes were
analyzed in pairs by blastp program (Altschul et al., 1997) and
homology between all genes in all unique pairs of gene rows were
determined according to E-value from blast results. Pairs of gene
rows with high diagonal homology were assigned as collinearity.
In the PILS1/PILS3/PILS4 group we found very high collinearity
between PILS3 and PILS4 (Figure 5A). These genes appear to be
products of very recent gene duplication. Between PILS1 and the
PILS3/PILS4 pair we also found high collinearity (Figures 5B,C)
and assume that these genes arose during full-genome duplication
at Brassicaceae family level (20 million years ago; Mya). Only very
weak or no collinearity was detectable between PILS5 and PILS7
(Figure 5D).
FIGURE 4 | Chromosomal distribution of Arabidopsis thaliana PILS
genes. A. thaliana PILS genes are found on chromosome 1, 2, and 5.
To further elaborate on the recent duplication of PILS3 and
PILS4, we analyzed the microevolutionary relationship between
PILS sequences of A. thaliana and A. lyrata (Figure S1 in Sup-
plementary Material). A. lyrata is the closest known relative of A.
thaliana and has a genome of eight chromosomes and six PILS
proteins (Van Bel et al., 2012). In contrast, A. thaliana has five
chromosomes and seven PILS proteins (Barbez et al., 2012; Van Bel
et al., 2012). It has been shown that the reduction of genome size in
A. thaliana is the result of chromosomes fusion that presumably
occurred about 5 Mya (Yogeeswaran et al., 2005). The phyloge-
netic analysis revealed that all six A. lyrata PILSes have highly
similar orthologs in A. thaliana, while AtPILS4 is a lineage-specific
gene (Figure S1 in Supplementary Material). This indicates that
AtPILS4 is a duplicated gene that has arisen after the separation of
A. thaliana from A. lyrata 5 Mya.
Next we addressed the sequence diversifications among the
A. thaliana PILS proteins and performed a ClustalW Multiple
sequence alignment (Larkin et al., 2007; Table 2; for sequence

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Feraru et al. PILS evolution and structural diversification

FIGURE 5 | Origin of PILS paralogs in Arabidopsis thaliana. (A–D) PILS genes show high collinearity between gene pairs PILS3/PILS4 (A), PILS1/PILS3 (B),
and PILS1/PILS4 (C). No or only very weak collinearity could be detected between PILS5 and PILS7 (D).

alignment see Figure S4 in Supplementary Material). PILS3/PILS4
showed the highest identity (82%), followed by PILS1/PILS3
(69%), PILS5/PILS7 (64%), and PILS1/PILS4 (61%; Table 2).
Interestingly, PILS2 and PILS6 showed sequence identity of
only 39% (Table 2). Taking together, the amino acid iden-
tity proposes that A. thaliana PILS proteins belong to three
subgroups: (i) PILS1/PILS3/PILS4, (ii) PILS5/PILS7, and (iii)
PILS2/PIL6.
PILS GENE REGULATION AND ORGANIZATION IN ARABIDOPSIS
THALIANA
To get further insight into the regulation of PILS activity, we ana-
lyzed in silico PILS gene organization and expression. A. thaliana
PILS gene transcripts organization is pretty well-conserved regard-
ing the number and size of the exons (Figure 6). PILS3 to PILS6
genes contain nine exons with more or less conserved size and
placements (Figure 6). In contrast, PILS1, PILS2, and PILS7 have

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Feraru et al. PILS evolution and structural diversification

Table 2 | Percentages* of Arabidopsis thaliana PILS amino acid Our results show that PILS intron/exon organization is largely
sequence identity calculated by ClustalW multiple sequence conserved among PILS orthologous (Figure 7). The variations of
alignment (Larkin et al., 2007). 1–2 more or less exons may be the result of insertions, deletions, or
both processes along the lineage evolution. The subfamily of PILS2
PILS7 genes is most particular, because they display single-exon genes in
31 PILS6 Angiosperms and Selaginella and 3-exons genes in Physcomitrella
31 64 PILS5 (Figure 7). Thus, PILS genes belong to two structural groups with
39 28 43 PILS4 1–3 exons (PILS2 orthologs and PILS genes from Ostreococcus and
82 41 31 43 PILS3 Micromonas) and 7–12 exons (all the other PILSes; Figure 7).
32 28 29 39 30 PILS2 PILS gene activity can be detected in all tissues of A. thaliana
28 69 61 40 29 42 PILS1 as shown by RT-PCR (Barbez et al., 2012) or by micro array-
PILS1 PILS2 PILS3 PILS4 PILS5 PILS6 PILS7 based online tools such as Genevestigator5 . PILS genes display
either relatively low (PILS1, PILS4, PILS7 ), medium (PILS6 ) or
*The identity percentages were calculated as the identities between two PILS
high (PILS2, PILS3, and PILS5) expression levels (see text foot-
sequences, divided by the length of the alignment.
note 5). PILS2-to-AtPILS6 are expressed in seedlings, leaves, and
flowers (Figure 8; Barbez et al., 2012; see text footnote 5). PILS4
displays the strongest expression in the rosette leaves (Barbez et al.,
2012). PILS6 transcripts are particularly abundant in the stem and
together with PILS5 in the cauline leaves and flowers, while PILS2
is highest in siliques (Barbez et al., 2012). Interestingly, some PILS
gene products were excluded from certain tissues. PILS1 was found
to be expressed only in flowers, PILS2 and PILS3 are not expressed
in the stem, PILS5 is absent in the rosette leaves, stem, and siliques,
while PILS6 and PILS7 were present in all plant organs but not
in siliques (Barbez et al., 2012). Except PILS1, all the other PILSes
were expressed in seedlings, with PILS5 and PILS2 having the
highest expression (Barbez et al., 2012). PILS2-to-PILS6 showed
expression in pollen with PILS5 being the most abundant (see text
footnote 5). Based on these evidences it seems that PILS genes
show specific and partially overlapping expression patterns in all
plant tissues.
Alternative splicing might furthermore contribute to the regu-
latory complexity and diversity for PILS gene activity. PILS3 and
PILS5 appear to bear two and four alternative transcripts, respec-
tively6 . In both cases the alternative gene splicing seems to occur
in the 50 region and may modulate PILS3 and PILS5 function.
However, the importance of PILS transcript splicing remains to be
demonstrated.
The pronounced differences in the expression levels and tis-
sue distributions might indicate that PILS-mediated regulation of
plant growth and development may be largely determined by gene
FIGURE 6 | Organization of Arabidopsis thaliana PILS genes. Schematic regulation.
intron/exon representation of A. thaliana PILS genes (Van Bel et al., 2012).
Exons and introns are depicted in blue and gray, respectively. 30 UTR and 50
PILS PROTEIN ORGANIZATION IN ARABIDOPSIS THALIANA
UTR are in green. Stars are showing exons of similar sizes (nucleotides):
red (80), green (125), blue (122), yellow (93), gray (101), and black (125).
The temporal and spatial regulation of PILS genes will give rise
to tissue specific distribution of distinct PILS proteins. Next we
analyzed predicted PILS protein organization and searched for
a divergent exon/intron structure. PILS1 has 12 exons, PILS7 bares domains to speculate on PILS function. PILS proteins range in
eight exons and PILS2 is even intron less. The size of exon number size from 390 (43 kDa; PILS3) to 472 (52 kDa; PILS1) amino acids.
2 (80 nucleotides), 3 (125 nucleotides), and 4 (122 nucleotides) However, the predicted protein topology is highly similar for all
is largely kept in AtPILS genes and encode for a highly conserved PILS proteins. PILS proteins are presumably characterized by two
region of the predicted transmembrane helices 2–4 (109 aa in hydrophobic transmembrane regions found at N- and C-termini
total). Also a C-terminal transmembrane domain seems to be (Figure 9A; Tusnády and Simon, 1998, 2001; Spyropoulos et al.,
encoded by the last exon (125 nucleotides) in almost all AtPILS 2004). The two transmembrane regions flank a short hydrophilic
genes (Figure 6).
Next, we analyzed the intron/exon organization of PILS genes 5 www.genevestigator.com

from algae, Physcomitrella, Selaginella, and several Angiosperms. 6 http://www.arabidopsis.org

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Feraru et al. PILS evolution and structural diversification

FIGURE 7 | Organization of PILS orthologs. Schematic intron/exon Ostreococcus lucimarinus (OL), Ostreococcus tauri (OT),
representation of PILS genes from Arabidopsis lyrata (AL), Arabidopsis Physcomitrella patens (PP), Selaginella moellendorffii (SM), and Volvox
thaliana (AT), Brachypodium distachyon (BD), Chlamydomonas carteri (VC; Van Bel et al., 2012). Exons are depicted in blue boxes,
reinhardtii (CR), Micromonas (MRCC299), Oryza sativa (OS), introns in gray lines.

region (loop) with a presumable cytosolic orientation (Figure 9A). among the PILS proteins (Figure 9). In contrast, the loop is less
Each hydrophobic region appears to be organized in five trans- conserved and is the most divergent part of the PILS sequences.
membrane helices that are very similar and highly conserved We assume that the transmembrane domains have central roles in

www.frontiersin.org October 2012 | Volume 3 | Article 227 | 9

Feraru et al.
FIGURE 8 | Transcription pattern of Arabidopsis thaliana PILS
genes. (A,B) Transcription of PILS genes in A. thaliana plant (A)
and root (B). Based on the RT-PCR data from individual organs
the putative carrier function, while the presumably cytosolic loop
might have rather regulatory functions.
PILS and PIN proteins share only 10–18% sequence identity
and belong to distinct protein families (Figure 3; Barbez et al.,
2012). However, the predicted topology of PILS proteins is rem-
iniscent to the predicted topology of PIN proteins (Krecek et al.,
2009) and allowed the identification of this novel putative auxin
carrier family (Barbez et al., 2012). Based on the hydrophilic loop
size, PIN proteins are sub-grouped into two subfamilies. The sub-
family of PIN1-type encompasses the PIN members with a long
hydrophilic loop and PM localization (PIN1–PIN4, PIN7), while
the subfamily of PIN5-type encompasses PIN5 and PIN8 that have
very short hydrophilic loops and ER localization. Although PIN6
shows a reduction of the loop size, PIN6 is often included in
the PIN1-type subfamily due to high sequence similarity in the
transmembrane regions (Krecek et al., 2009). However, it is also
localized to the ER in transient localization studies (Mravec et al.,
2009).
Similarly to PIN proteins, PILS family members are character-
ized by the presence of the Interpro auxin carrier domain. This
Interpro domain is relatively long and spans almost the whole
length of the PILS protein and, hence, it is difficult to ascertain
functional residues within the “domain”.
Nothing is yet known about the post-translational modification
of PILS proteins but generic phosphorylation sites (non-kinase-
specific, such as serine, threonine, and tyrosine), kinase-specific
phosphorylation sites and isoform variations could be predicted
Frontiers in Plant Science | Plant Traffic and Transport
PILS evolution and structural diversification
published in Barbez et al. (2012). (A) As predicted by online server
Arabidopsis eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/
efpWeb.cgi); (B).
for PILS proteins by available online servers such as NetPhos
(Blom et al., 1999) and NetPhosK (Blom et al., 2004). Interest-
ingly, according with the number of the predicted serine, thre-
onine, and tyrosine phosphorylation sites, PILS proteins can be
grouped into three classes: (i) less than 10 (PILS5 and PILS7), (ii)
between 10 and 15 (PILS2 and PILS6), and (iii) more than 15
(PILS1, PILS3, and PILS4). This finding may indicate the func-
tional diversification among the PILS members and may suggest
that different phosphorylation-based mechanisms are required for
the regulation of PILS activity.
DISCUSSION
Auxin has pronounced importance for the plant development.
Recent research shed light on a particular link between intracel-
lular auxin transport processes and auxin metabolism (Mravec
et al., 2009; Barbez et al., 2012; Bosco et al., 2012; Ding et al.,
2012). Here, we report in silico analyses of PILS putative auxin
flux facilitator sequences from A. thaliana and revealed certain
features that might be functionally important for PILS activity.
The phylogenetic analysis of PILS sequences revealed that four
Physcomitrella PILSes are found in Clade II, while only one is found
in Clade III (Figure 2). Moreover, two Selaginella PILSes are found
in each, Clade III and Clade II-PILS2 subclade, while four paralogs
are found in the Clade II-PILS6 subclade (Figure 2). This, together
with the distribution of the Brachypodium, Oryza, and Arabidopsis
PILS sequences, indicates that the initial PILS divergence occurred
in two separate clades already at the level of Bryophytes. We do not
October 2012 | Volume 3 | Article 227 | 10
Feraru et al.
FIGURE 9 | Structure of Arabidopsis thaliana PILS proteins. (A)
Predicted topology of A. thaliana PILS5 protein. The prediction was
done by HMMTOP 2.0 (Tusnády and Simon, 1998, 2001) and visualized
by TMRPres2D (Spyropoulos et al., 2004). Conserved amino acids in all
seven PILS proteins are marked in red. (B) Sequence logos generated
know if PILSes are present in the genome of Rhodophytes, but we
can speculate that Clade II- and Clade III-PILSes may have origi-
nated before land plant evolution at the level of Streptophytes, as
these algae are direct ancestors of land plants. Moreover, Clade II
presumably diverged before or during the origin of Embryophytes,
because this clade is already diversified in PILS2- and PILS6-like
subclades in mosses (Figure 2). Clade III particularly expanded
during higher plant evolution (Figure 2; Figure S1 in Supple-
mentary Material). This clade is divided in PILS1/PILS3/PILS4
and PILS5/PILS7 subclades (Figure 2; Figure S1 in Supplementary
Material). We could not estimate when these subclades emerged
because PILS sequences from conifers and ferns are either incom-
plete (only ESTs available) or not available. More than 30000 ESTs
www.frontiersin.org
PILS evolution and structural diversification
by WebLogo (Schneider and Stephens, 1990) representing a ClustalW
multiple sequence alignment (Larkin et al., 2007) of 109 amino acids
from N-terminal region of A. thaliana PILS proteins (exons 2–4). Note
the PILS sequence conservation at the highest, single symbol
positions.
derived from gametophyte of fern Adiantum can be found on
NCBI but we could not identify any PILS sequence which indicates
that PILSes might be not transcribed in gametophyte.
Combining the gene and protein analyses, AtPILS4 is likely to
be a recent duplication of AtPILS3, because they show very high
amino acid identity (Table 2), strong gene collinearity (Figure 5A),
and no particular PILS4 orthologs could be identified in the
genomes of the other sequenced species. PILS3/PILS4 seem to be
originally derived from PILS1 (69% amino acid identity; Table 2).
Accordingly, by analyzing the amino acid sequence similarities and
the PILS phylogeny, we can conclude that from seven PILSes in A.
thaliana genome, six in Oryza sativa ssp japonica and eight in
Brachypodium distachyon genome only four are true orthologs.
October 2012 | Volume 3 | Article 227 | 11
Feraru et al. PILS evolution and structural diversification

The other PILS members presumably represent lineage-specific
duplications that occurred after the separation of the dicots and
monocots about 200–250 Mya.
The existence of PILS2 as a single-exon gene in most species is
intriguing since single-exon genes are rather typical for prokary-
otes. However, single-exon or intronless genes are present in
eukaryotic genomes (Sakharkar et al., 2004) and can have many
origins, but could pinpoint the relatedness to a prokaryotic
gene (Zou et al., 2011). However, moss PILS2 orthologs display
intronexon structure (Figure 7) and might suggest that PILS2
genes lost the intron structure during evolution.
Our findings might highlight certain functional diversifications
among PILS proteins. Notably, PILS2 and PILS5 have only 29%
amino acid sequence identities (Table 2), display very diverged
gene organization (Figure 6), and belong to diverse evolutionary
sub clades (Figure 2). However, their gene regulation and function
seem to be highly similar in Arabidopsis, because PILS2 and PILS5
have overlapping expression pattern in the root transition zone
and redundantly control seedling growth and development (Bar-
bez et al., 2012). Therefore, defined research is needed to evaluate
the functional importance of the distinct features of the respective
PILS genes and PILS proteins.
Figure S1 | Molecular phylogenetic analysis of PILS proteins. The diagram
shows an extended phylogentic tree of PILS proteins with collapsed branches
for algae, Physcomitrella, and Selaginella. Note the high diversification of PILSes
in Medicago and Populus. Because of incomplete sequences some of the
PILSes were eliminated. The evolutionary history was inferred by using the
Maximum Likelihood method based on the Data specific model (Nei and Kumar,
2000). The tree with the highest log likelihood (−55875.7936) is shown. The
percentage of trees in which the associated taxa clustered together is shown
above the branches. Initial tree(s) for the heuristic search were obtained
automatically as follows. When the number of common sites was <100 or less
than one fourth of the total number of sites, the maximum parsimony method
was used; otherwise BIONJ method with MCL distance matrix was used. A
discrete Gamma distribution was used to model evolutionary rate differences
among sites [five categories (+G, parameter = 2.6899)]. The rate variation model
allowed for some sites to be evolutionarily invariable ([+I], 3.7299% sites). The
tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. The analysis involved 75 nucleotide sequences. All
positions with less than 0% site coverage were eliminated. That is, fewer than
100% alignment gaps, missing data, and ambiguous bases were allowed at any
position. There were a total of 1113 positions in the final dataset. Evolutionary
analyses were conducted in MEGA5 (Tamura et al., 2011).
Figure S2 | Alignment of PILS amino acid sequences. The multiple amino
acid alignment of PILSes was generated by using Muscle in MEGA5 software
(Tamura et al., 2011). This alignment was generated for the phylogenetic analysis
presented in the Supplementary Figure 1. The alignment for the smaller tree
presented in the Figure 2 is similar but with less sequences.

ACKNOWLEDGMENTS
We are indebted to the Vienna Science and Technology Fund
(WWTF; to Elena Feraru, Mugurel I. Feraru, Jürgen Kleine-
Vehn), Czech Science Foundation Grant GAP305/11/2476 (to Jan
Petrášek) and Charles University in Prague, SVV 265203/2012 (to
Jan Petrášek and Stanislav Vosolsobě). We thank Elke Barbez for
helpful discussions.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at
http://www.frontiersin.org/Plant_Traffic_and_Transport/10.3389/
fpls.2012.00227/abstract
Figure S3 | Alignment of PILS and PIN amino acid sequences. The multiple
alignment was generated by using Muscle in MEGA5 software (Tamura et al.,
2011).
Figure S4 | Alignment of Arabidopsis thaliana PILS amino acid sequences.
A multiple sequence alignment generated by ClustalW server (Larkin et al.,
2007) of the seven PILSes is shown. Amino acids are color coded: red (small,
hydrophobic, aromatic, not Y), blue (acidic); magenta (basic), green (hydroxyl,
amine, amide, basic), gray (others). “∗,” Identical amino acids; “:,” conserved
substitutions (same color group); “.,” semi-conserved substitution (similar
shapes).
Table S1 | Sequence information.

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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.
Received: 12 September 2012; accepted:
21 September 2012; published online: 12
October 2012.
Citation: Feraru E, Vosolsobě S, Fer-
aru MI, Petrášek J and Kleine-Vehn J
(2012) Evolution and structural diversi-
fication of PILS putative auxin carriers
in plants. Front. Plant Sci. 3:227. doi:
10.3389/fpls.2012.00227
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